Supplementary Materials and Methods
Colony forming assay
Cells were treated with the drug for 24 h and at the end of drug treatment, cells were
washed with 1 x PBS, trypisinized, and counted. For every treatment, 5,000 cells were
re-plated onto a 100mm dish in complete tissue culture medium. Re-plated cells were
left to form colonies over a period of 10 to 14 days in a humidified incubator at 37 oC
with 5% CO2, supplemented with complete medium every 3-5 days. Colonies formed
were assessed by staining cells with crystal violet.
Isolation of membrane lipid rafts by sucrose density gradient ultracentrifugation
A549 WT cells (2x106) or R1 cells (4x106) were seeded in 100mm petri dishes 2 days
before the lipid raft extractions. Cells were harvested and washed with ice-cold
1xPBS before incubating in 500ml of HEPES buffer (25 mM HEPES, 150 mM NaCl,
pH7.4 containing 1% Triton X-100) and supplemented with the protease inhibitors
(1mM PMSF, 10 μg/ml Aprotinin, 20 μg/ml Pepstatin A) and phosphatase inhibitors
(1mM NaF, 1 mM Na3VO4) on ice for 10 min. Lysates were then subjected to 1 round
of freeze-thaw action before homogenizing with a Dounce homogenizer for 20
strokes. The homogenized lysates were centrifuged at 500g for 10 min at 4°C to
remove debris and the supernatants were subjected to 10 passes through a 27G
syringe. This was followed by sonication (3 sets of 10 sec pulses at 40V) and protein
quantification to standardize the amount of protein used for subsequent
ultracentrifugation. The samples were then mixed with an equal volume of 80%
sucrose (w/v) in HEPES buffer, transferred to a centrifuge tube and carefully overlaid
with 2ml of 30% sucrose and 2ml of 5% sucrose solutions. The sucrose gradient was
centrifuged for 21 h at 4ºC in a Beckman rotor at 32,000 rpm and nine 0.5 ml fractions
were collected from top to bottom (named 1-9). The fractions were subjected to SDSPAGE and Western blotting and lipid rafts are validated by the presence of lipid raft
markers, caveolin-1 and flotillin.
Detection of phosphatidylserine externalization by Annexin V staining
Following treatment, cells were harvested and pelleted down by centrifugation at
2,000 rpm. Cells were washed in 1X PBS, then once in 1X Binding Buffer. Cells were
then re-suspended in 1X Binding Buffer (diluted from 10X buffer: 0.1M Hepes, 1.4M
NaCl, and 25 mM CaCl2 with dH2O at pH 7.4) and stained with FITC-conjugated
Annexin V (BD Biosciences; San Jose, CA, USA) for 15 min at room temperature.
Subsequently, cells were washed twice in 1X Binding Buffer and re-suspended in
200ul of 1x binding buffer for flow cytometry (Beckman Coulter Inc., Sunnyvale,
CA) using an excitation wavelength of 488 and emission at 525nm. Data for 10,000
events were collected and analyzed by Summit 4.3 software.
Determination of caspase activity
Caspase activation was determined using synthetic fluorogenic oligopeptide
substrates with the C-terminal aspartic acid modified with 7-amino 4 trifluoromethyl
coumarin (AFC). Cells were harvested on ice at the end of different time points and
lysed with chilled 1x Cell Lysis Buffer (BD Pharmingen; San Diego, CA). Protein
concentration was quantified using the Coomassie Blue reagent. 5-10ug of cell lysates
were added to equal volumes of 2X Reaction Buffer (20mM HEPES, 4mM EDTA,
10mM DTT and 3mM MgCl2 pH 7.4) and 1uM of respective caspase substrates was
added on a 96-well plate; AcDEVD-AFC for caspase 3, Ac-VEID-AFC for caspase 6,
Ac-IETD-AFC for caspase 8 and Ac-LEHD-AFC for caspase 9. Samples were
incubated at 37°C for 1 hour, after which caspase activity was determined by
measuring the relative AFC fluorescence with TECAN spectrophotometer (Tecan
Trading AG, Switzerland) with excitation at 400nm and emission at 505nm. Protein
concentration of each sample was determined using the Coomassie Blue reagent and
caspase activity was normalized with protein amount and expressed as relative
fluorescence unit (RFU)/μg protein.
Analysis of DR4 and DR5 surface expression
A549 WT, R1 and R2 cells were seeded in 6-well plates for 24 h and harvested and
washed with FACS buffer (1X PBS + 0.5% FBS) after detachment of adherent cells
with EDTA. Cells were then stained with the phycoerythrin (PE)-conjugated mouse
monoclonal anti-human DR4 or DR5 for 45 min at 4oC according to manufacturer’s
instructions before washing and resuspension in FACS buffer for flow cytometry
(CyAn ADP, Beckman Coulter, USA) using an excitation wavelength of 488. PE-
conjugated mouse IgG2b was used as an isotype control and the obtained events were
analysed by Summit 4.3 software.
Confocal microscopy for lipid raft localization of DR4 and caspase-8
A549 WT cells and R1 cells were plated in a 12-well plate on polylysine-coated
coverslips. Cells were washed twice with 1X PBS and fixed with 2%
paraformaldehyde in 1X PBS for 15min at room temperature. Next, cells were
washed three times with ice cold 1X PBS and subjected to permeabilization with
50M of digitonin in 1X PBS for 15min at room temperature. Cells were then washed
three times with ice cold 1X PBS followed by 1h blocking with 5% BSA.
Subsequently, cells were incubated with either mouse anti-DR4 or mouse anticaspase-8 and rabbit anti-caveolin-1 in 1X PBS with 5% BSA (1:250 dilution for antiDR-4 and anti-caspase-8, 1:500 dilution for anti-caveolin-1) for 2h. After that, they
were washed three times with 1X PBS in 5% BSA followed by 1h incubation with
1:500 dilution for Alexafluor-488 anti-mouse secondary antibody (Invitrogen) and
1:500 dilution for Alexafluor-568 anti-rabbit secondary antibody in 1X PBS with 5%
BSA. Cells were similarly washed three times with 1X PBS before the coverslips
were mounted on glass slides and imaged with Olympus Fluoview FV1000 confocal
microscope and images were analyzed using Olympus Fluoview 1.7 viewer.